PROJECT SUMMARY Despite extensive knowledge of genes required for gametogenesis, the causes of most human infertilities are unknown. It is thought that about half of cases have a genetic basis, but it remains problematic to definitively identify the exact genetic lesion(s) that may be responsible in a given person, even with genome sequence information. Given the indications that certain genetically-based causes of infertility are associated with comorbidities, and the high incidence of infertility in the population, it would be of substantial clinical impact to make progress towards reliably identifying molecular causes of infertility. The roots of non-genetic causes are even more difficult to ascertain, but epigenetic alterations may underlie certain types of infertility, particularly in males where such alterations may occur in spermatogonial stem cells (SSCs). Project II has 3 Specific Aims that seek to address these important issues in the field. Aim 1 is to functionally validate candidate genetic variants identified from infertility patient cohorts and families in Project 1. This will be done by modeling the variants in CRISPR-modified mice, then phenotyping the mice for not only reproductive phenotypes, but also comorbidities that may exist in the corresponding patients, such as obesity, cancer susceptibility, or cardiovascular disease. Aim 2 is to characterize mutations that cause infertility or subfertility phenotypes in mice, and which may have comorbidities that have yet to be recognized. An emphasis will be on genes that are expressed in somatic tissues in addition to the testis. Aim 3 employs a novel approach to identify epigenetic alterations that impact SSCs, potentially leading to Sertoli Cell Only syndrome (SCOS) and non-reproductive phenotypes. This approach involves CRISPR inhibition and activation technologies to conduct a screen of known epigenetic modification genes that impact the efficiency somatic cell reprogramming and SSC maintenance. In sum, this project uses diverse, cutting-edge strategies to improve the accuracy and discovery of genetic and epigenetic causes of human male infertility and related comorbidities.